Integrated device for real time quantitative PCR

Abstract

A method for real-time quantitative detection of single-type, target nucleic acid sequences amplified using a PCR in a microwell, comprising introducing in the microwell a sample comprising target nucleic acid sequences, magnetic primers, and labelling probes; performing an amplification cycle to form labelled amplicons; attracting the magnetic primers to a surface through a magnetic field to form a layer including labelled amplification products and free magnetic primers; and detecting the labelled amplification products in the layer with a surface-specific reading method.

The invention claimed is: 
     
       1. A method comprising:
 forming magnetic amplification products by amplifying a target nucleic acid in a sample with magnetic primers that are in a microwell having a bottom surface and first walls that extend away from the bottom surface; and 
 forming a multilayer of the magnetic amplification products in a recess in the bottom surface of the microwell, the recess having second walls spaced inwardly from the first walls by attracting and laterally confining the magnetic primers to the recess using a magnetic field source positioned at the recess. 
 
     
     
       2. The method of  claim 1 , further comprising quantitatively detecting the magnetic amplification products by measuring a probe in the microwell that is hybridized to the multilayer by using a spatially discriminating detection device. 
     
     
       3. The method of  claim 1 , further comprising quantitatively detecting the magnetic amplification products by measuring a probe in the microwell that is hybridized to the multilayer by using a confocal detector or microscope. 
     
     
       4. The method of  claim 1 , wherein the magnetic field source having a first dimension in a first direction and the recess has a second dimension in the first direction, the second dimension being greater than or equal to the first dimension. 
     
     
       5. The method of  claim 1 , wherein the magnetic primers include primers coupled to paramagnetic beads having diameters less than 50 nanometers (nm). 
     
     
       6. The method of  claim 5 , wherein the paramagnetic beads are single crystals. 
     
     
       7. A method comprising:
 contacting magnetic primers with a labelled probe and a sample, which includes a target nucleic acid, in a microwell having a bottom surface; 
 forming first magnetic amplification products by performing at least one amplification cycle; 
 forming a first multilayer of the first magnetic amplification products by attracting and laterally confining the magnetic primers to a recessed central area of the bottom surface using a magnetic field source extending adjacent to the recessed central area; and 
 quantitatively detecting the first magnetic amplification products by measuring the probe that is hybridized to the first multilayer. 
 
     
     
       8. The method of  claim 7 , wherein a width of the recessed central area is substantially the same as a width of the magnetic field source. 
     
     
       9. The method of  claim 7 , wherein measuring the probe includes measuring the probe using a confocal detector or microscope. 
     
     
       10. The method of  claim 7 , wherein a ratio of the width of the magnetic field source to the width of the bottom surface ranges from 1:10 to 1:5. 
     
     
       11. The method of  claim 7 , wherein quantitatively detecting the first magnetic amplification products includes measuring the probe during the at least one amplification cycle. 
     
     
       12. The method of  claim 7 , further including:
 releasing the first multilayer; 
 forming second magnetic amplification products by performing at least one additional amplification cycle; 
 forming a second multilayer of the first and second magnetic amplification products in the recessed central area by attracting and laterally confining the magnetic primers to the recessed central area, the probe being hybridized to the second multilayer; and 
 quantitatively detecting the first and second magnetic amplification products by measuring the probe. 
 
     
     
       13. A method comprising:
 contacting magnetic primers with a labelled probe and a sample that includes a target nucleic acid in a microwell having a bottom surface including a recess in a central portion, the recess having sidewalls and a width that is less than a width of the bottom surface; 
 forming magnetic amplification products by amplifying the target nucleic acid; 
 forming a multilayer of the magnetic amplification products in the recess by attracting the magnetic primers to the central portion using a magnetic field source extending adjacent to the central portion, the labelled probe being hybridized to the multilayer; and 
 quantitatively detecting the magnetic amplification products by measuring the labelled probe. 
 
     
     
       14. The method of  claim 13 , wherein quantitatively detecting the magnetic amplification products includes measuring the labelled probe using a spatially discriminating detection device. 
     
     
       15. The method of  claim 14 , wherein the spatially discriminating detection device has a focal plane focused at a central region of the multilayer. 
     
     
       16. The method of  claim 13 , wherein the measuring the labelled probe includes measuring the labelled probe using a confocal detector or microscope. 
     
     
       17. The method of  claim 13 , wherein the amplifying the target nucleic acid includes performing at least one amplification cycle, and wherein quantitatively detecting the first magnetic amplification products includes measuring the labelled probe during the at least one amplification cycle. 
     
     
       18. The method of  claim 13 , wherein a ratio of the width of the magnetic field source to the width of the bottom surface ranging from 1:10 to 1:5. 
     
     
       19. The method of  claim 13 , wherein the width of the recess is substantially the same as a width of the magnetic field source. 
     
     
       20. The method of  claim 13 , wherein the magnetic primers include primers coupled to paramagnetic beads having diameters less than 50 nm.